Derivatisation

Derivatisation is carried out to make molecules more volatile, more easily separated, more symmetrical in peak shape, more easy to detect (possibly at lower levels), and less likely to undergo any thermal degradation. There is a vast literature on derivatisation. The most common derivatives for surfactant analysis are esterification of fatty acids and silylation of hydroxyl compounds. 

In the HPLC of drugs and poisons, derivatisation reactions, although sometimes used to stabilise an analyte, are seldom needed to achieve satisfactory chromatography except when performed to permit the separation of enantiomers for example Srinivas and Igwemezie list a number of derivatisation reagents that have been used to facilitate enantiomer separations by HPLC-ECD. However, derivative formation is chiefly used to enhance the selectivity and sensitivity of detection. Ruorescent derivatives are the most popular, but derivatisation has also been used to enhance UV or EC detection characteristics.  

Many reagents can be used in either pre- or post-column mode. Primary amines may be derivatised using OPA in the presence of a thiol (2-mercaptoethanol, A-acetylcysteine, 3-mercaptopropionate, -butylthiol) or sodium sulfite and by other methods developed primarily for amino acid analysis. EC oxidation or, more usually, fluorescence detection following post-column reaction with OPA/2-mercaptoethanol may be used for primary aliphatic amines, although some compounds, such as tocainide, do not react under all conditions. The OPA/2-mercapto-ethanol derivative of histamine can be oxidised at a potential of -1-0.4 V vs Ag/AgCl, whereas histamine itself requires more than +1.2 The derivative 

3-Naphthalenedialdehyde (NDA) in the presence of cyanide ion is an alternative to OPA. The methodology was developed to produce fluorescent derivatives, but products derivatised at more than one site may not be fluorescent due to quenching. However, they are oxidisable at approximately +0.75 V V5 Ag/AgCl. Sherwood et al derivatised over 40 a-amino acids using phenyl-isothiocyanate, a particularly useful reagent as imino acids, such as proline, also react. The derivatives were analysed on an ODS-modified silica column with 

The extent to which derivatisation will be used to enhance ED in future is uncertain. Several of the examples cited above used novel reagents. Unless these reagents are made easily available, it is difficult to believe that these techniques 

Post-column photolysis to give electroactive products has been used in the measurement of trichothecene mycotoxins, such as deoxynivalenol (vomitoxin) in 

In liquid chromatography, in contrast to gas chromatography [see Section 9.2(2)], derivatives are almost invariably prepared to enhance the response of a particular detector to the substance of analytical interest. For example, with compounds lacking an ultraviolet chromophore in the 254 nm region but having a reactive functional group, derivatisation provides a means of introducing into the molecule a chromophore suitable for its detection. Derivative preparation can be carried out either prior to the separation (pre-column derivatisation) or afterwards (post-column derivatisation). The most commonly used techniques are pre-column off-line and post-column on-line derivatisation. 

Pre-column off-line derivatisation requires no modification to the instrument and, compared with the post-column techniques, imposes fewer limitations on the reaction conditions. Disadvantages are that the presence of excess reagent and by-products may interfere with the separation, whilst the group introduced into the molecules may change the chromatographic properties of the sample. 

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

Reagents which form a derivative that strongly absorbs UV/visible radiation are called chromatags an example is the reagent ninhydrin, commonly used to obtain derivatives of amino acids which show absorption at about 570 nm. Derivatisation for fluorescence detectors is based on the reaction of non-fluorescent reagent molecules (fluorotags) with solutes to form fluorescent 

The reader is recommended to consult the relevant textbooks56 (see also Bibliography, Section 9.10) for a more comprehensive account of chemical derivatisation in liquid chromatography. 

The use of sodium tetraethylborate overcomes the problem of hydrolytic instability of the Grignard reagents, allowing ethylation to be carried out in an aqueous medium. This reaction has been employed for the analysis of alkyl-Pb-, Sn- and Hg-species (Ashby and Craig, 1989). Aqueous phase ethylation is growing in popularity because extraction into an organic phase is avoided and one source 

The separation of chemical species of elements can only be performed by techniques which do not destroy the chemical forms, e.g. possible degradation of the species upon heating of the column. Separation is generally performed after extraction and suitable clean-up of the extract. The most popular methods applied nowadays in speciation studies are GC (capillary or packed), HPLC and cold-trapping. 

To date, capillary GC is certainly the separation method which is the most frequently used for the determination of chemical species. Packed GC columns have been progressively abandoned owing to their poor resolution. 

Cold-trapping has been used successfully for the determination of alkyl-Sn and Se compounds, Pb and some As compounds (Donard, 1987). The technique presents the advantages of both concentrating the species and sequentially separating them according to their specific volatility. One drawback of this method is that only volatile forms of elements (hydrides, ethylated or methylated forms) may be separated, requiring a derivatisation step other molecules of low 

There are four principal ways of denoting the mass of an ion in the mass spectrum  

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Brust M ef a/1994 Synthesis of thiol-derivatised gold nanopartioles in a two-phase liquid-liquid system J. Chem. Soc. Chem. Comm. 801... 

Liquid Ghromatography/Mass Spectrometry. Increased use of Hquid chromatography/mass spectrometry (Ic/ms) for stmctural identification and trace analysis has become apparent. Thermospray Ic/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions (74). Five compounds resulting from the reaction of phenyUsocyanate and the reaction medium were identified two from a reaction between phenyl isocyanate and methanol, two from the reaction between phenyl isocyanate and water, and one from the polymerisation of phenyl isocyanate. There were also two reports of derivatisation to enhance either the response or stmctural information from thermospray Ic/ms for linoleic acid hpoxygenase metabohtes (75) and for cortisol (76). 

Analysis of substances (this is a key process in the identification of new or known chemical and biological substances. Methods of analysis include spectroscopic methods, derivatisation and sequencing meth s). 

There is a large range of resins available for SPOS. These resins are derivatised polymer supports with a range of linkers. The roles of linkers are (i) to provide point(s) of attachment for the tethered molecule, akin to a solid supported protecting group(s), (ii) to provide distance from the polymeric backbone in order to minimise interactions with the backbone, (iii) to enable cleavage of product molecules under conditions compatible with the stability of the molecules and the reaction conditions employed for chemical transformations. Hence in order to... 

The AMAPs (2-[ arylmethyl amino]-l,3-propanediols) are a class of planar polycyclic aromatic derivatives, which contain polar side-chains. They are known to be DNA intercalators and possess broad spectrum antitumour activity. An approach to C-radiolabelled AMAP derivative 40 used the Bucherer reaction as an initial starting reaction. 2-Naphthol was reacted with 4-bromophenylhydrazine 38 in the presence of sodium metabisulfite and HCl to afford 39. Subsequent derivatisation of 39 afforded 40. 

Various types of derivatisation have now been developed for both gas and liquid chromatography. For more detailed information regarding the choice of a suitable derivative for a particular analytical problem, the appropriate works of reference should be consulted.62,63... 

The purpose of the experiment is to illustrate the application of derivatisation in the analysis of sugar and related substances by gas-liquid chromatography. The silylation method described is an almost universal derivatisation procedure for carbohydrate analysis by GC.79... 

J F Lawrence and R W Frei, Chemical Derivatisation in Liquid Chromatography, Elsevier, Amsterdam, 1976... 

D Knapp (Ed), Handbook of Analytical Derivatisation Reactions, Wiley, New York, 1979... 

Deposit character of, (eg) 509 Deposition completeness of, 507 potentials of metals, 508 Derivative spectrofluorimetry 733 Derivative spectrophotometry 668 Derivatisation in chromatography, gas, 236 liquid, 228 in fluorimetry, 735 Desiccants 99 Desiccators 98 cooling of crucible in, 100 desiccants for, 99 Scheibler type, 99 uses of, 100 vacuum, 100... 

Gamma, of a photographic emulsion 769 Gas chromatography 235 apparatus for, 235 column packing for, 238 derivatisation in, 236 detectors for, 240 elemental analysis by, 247 of metal chelates 237, 248 pyrolysis, 237... 

Amylopectin is the polymeric component of starch and consists mainly of glucose units joined at the 1,4-positions. Relative molar mass tends to be very high, e.g. between 7 and 70 million. A variety of modified starches are used commercially which are produced by derivatisation to give materials such as ethanoates (acetates), phosphates, and hydroxyalkyl ethers. Modified and unmodified starches are used in approximately equal tonnages, mainly in papermaking, paper coatings, paper adhesives, textile sizes, and food thickeners. 

Transformation of bromocriptine free base 2 into water soluble salt -mesylate, is the only way to obtain a suitable therapeutical form. Crystallization of mesylate using alcohol as a solvent in the presence of excess of strong acid, e.g. methanesulphonic acid can induce formation of 12 -0-alkyl-derivative 2. Until now this derivatisation of ergot molecule has been practically unknown. In continuation we developed the preparative method for obtaining these compounds, (using tetrafluoroboric acid as a catalyst) (ref. 20). 

A similar procedure was adopted for synthesis of nanoparticles of cellulose (CelNPs). The polysaccharide nanoparticles were derivatised under ambient conditions to obtain nanosized hydrophobic derivatives. The challenge here is to maintain the nanosize even after derivatisation due to which less vigorous conditions are preferred. A schematic synthesis of acetyl and isocyanate modified derivatives of starch nanoparticles (SNPs) is shown in scheme 3. The organic modification was confirmed from X-ray diffraction (XRD) pattern which revealed that A- style crystallinity of starch nanoparticles (SNPs) was destroyed and new peaks emerged on derivatisation. FT-IR spectra of acetylated derivatives however showed the presence of peak at 3400 cm- due to -OH stretching indicating that the substitution is not complete. 

The way that Pn peptides self-assemble is important for polypeptides and proteins amyloid-type structures are believed to have the same core stmcture, and in fact the propensity to self-assemble in this manner is hypothesized to be a general property of polypeptides [52]. It is therefore unsurprising that systems featuring this motif are common. In recent years, a push towards the use of peptide-based self-assembled materials has led to increasing interest in extending their functionality by derivatising them. 

Fig. 16 (a) Sequences of peptides pi6 and B2x-pi6. (b) Biotin-derivatised colibrils binding to a functionalised streptavidin with a functional group. Reproduced by permission of The Royal Society of Qiemistry from Kodama et al. [56]... 

In this instance, adamantane was present to promote interaction of peptides through its hydrophobicity, but its attachment did not hinder fibril formation. It might be possible to chemically or biologically derivatise this group before being introduced to the peptide, or to select another hydrophobic component that could be suitably modified and attached to the peptide. This research also highlights the feasibility of creating peptide arrays comprised of several different sequences. 

Lyim and co-workers carried out smdies of the Ap(io 35) peptide - derived from residues 10-35 of the p-amyloid responsible for Alzheimer s disease - which forms fibrils composed of parallel p-sheets [62]. The peptide was compared to its C-terminal PEG-derivatised analogue. TEM experiments showed that both formed fibrils [63] (Fig. 21) but the uranyl acetate stain was not found inside the peptide-PEG fibrils, indicating that PEG was at the outer edge of the fibril. 

Fig. 23 Sequences of various peptides and PEG-derivatised peptides. Reproduced with permission from Collier and Messersmith [68], Copyright Wiley-VCH...

Amino acid composition was analysed as described by Barkholt and Jensen (6). Cysteine was determined after derivatisation with 3,3 -dithiopropionic acid. Tryptophan was not determined. 

The hydrosi(ly)lations of alkenes and alkynes are very important catalytic processes for the synthesis of alkyl- and alkenyl-silanes, respectively, which can be further transformed into aldehydes, ketones or alcohols by estabhshed stoichiometric organic transformations, or used as nucleophiles in cross-coupling reactions. Hydrosilylation is also used for the derivatisation of Si containing polymers. The drawbacks of the most widespread hydrosilylation catalysts [the Speier s system, H PtCl/PrOH, and Karstedt s complex [Pt2(divinyl-disiloxane)3] include the formation of side-products, in addition to the desired anh-Markovnikov Si-H addition product. In the hydrosilylation of alkynes, formation of di-silanes (by competing further reaction of the product alkenyl-silane) and of geometrical isomers (a-isomer from the Markovnikov addition and Z-p and -P from the anh-Markovnikov addition. Scheme 2.6) are also possible. 

This is the first example of a reaction for which the presence of a chelating ligand was observed to facilitate rather than retard metal-catalysed epoxidation (Gao et al., 1987). It was found that the use of molecular sieves greatly improves this process by removing minute amounts of water present in the reaction medium. Water was found to deactivate the catalyst. All these developments led to an improved catalytic version that allows a five-fold increased substrate concentration relative to the stoichiometric method. Sensitive water-soluble, optically active glycidols can be prepared in an efficient manner by an in situ derivatisation. This epoxidation method appears to be competitive with enzyme-catalysed processes and was applied in 1981 for the commercial production of the gypsy moth pheromone, (-1-) disparlure, used for insect control (Eqn. (25)). 

Steinbrecher, U.P. (1987). Oxidation of human low density lipoprotein results in derivatisation of lysine residues of apolipoprotein B by lipid peroxidation decomposition products. J. Biol. Chem. 262, 3603-3608. 

Chassaigne H, and Lobinski R (1998) Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS. Analyst 123 2125- 2130. Chemosphere (1999) Special issue - Sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39 1037-1224. 

An SFE instrument can be designed as a single standalone instrument performing a range of manual processes extraction, fractionation, concentration, solvent exchange, reconstitution and derivatisation. 

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